Overview

Also Known As: TRE-hM3Dq

These TRE-hM3Dq transgenic mice may be bred to generate Tet-Off/Tet-On mutant mice with conditional (inducible/reversible) expression of hM3Dq; a mutant G protein-coupled receptor (GPCR) that activates the canonical G_q pathway specifically following administration of the pharmacologically inert molecule clozapine-N-oxide (CNO). TRE-hM3Dq transgenic mice may be useful for chemogenetic/pharmacogenetic applications to study receptor-specific functions or general downstream cellular signaling emanating from the activated GPCR.

Donating Investigator

Bryan L Roth, University of North Carolina at Chapel Hill

Genetic overview

Genetic Background	Generation

Tg(tetO-CHRM3*)1Blr

Allele Type
Transgenic (Inducible, Inserted expressed sequence)

View Genetics

Research Applications

Research Tools
Neurobiology Research
Diabetes and Obesity Research
Immunology, Inflammation and Autoimmunity Research
Cell Biology Research
Cancer Research

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Base Price

Starting at:

$2,595.00 Domestic price Cryo Recovery

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Details

Detailed Description

Hemizygous TRE-hM3Dq transgenic mice are viable and fertile, with no reported phenotypic abnormalities. The TRE-hM3Dq transgene has a modified Tet response element (TRE or tetO) upstream of the mutant G protein-coupled receptor, hM3Dq (a human muscarinic 3 receptor with two amino acid substitutions (Y149C^3.33/A239G^5.46) that abolish receptor affinity for the native ligand, acetylcholine (ACh), but allow receptor binding and subsequent activation by the small pharmacologically inert molecule clozapine-N-oxide (CNO)). When bred with another mouse expressing tetracycline-controlled transactivator protein (tTA) or reverse tetracycline-controlled transactivator protein (rtTA), hM3Dq expression in the resulting double mutant offspring can be regulated with tetracycline or its analog doxycycline (dox). As designed, TRE-hM3Dq transgenic mice have no reported levels of hM3Dq activity in tTA(+dox) or rtTA(-dox) cell types. In TRE-hM3Dq transgenic tTA(-dox) or TRE-hM3Dq transgenic rtTA(+dox) cells types, hM3Dq expression results in activation of the canonical G_q pathway only following administration of CNO.

These TRE-hM3Dq transgenic mice may be useful to study receptor-specific functions or general downstream cellular signaling emanating from the activated G protein-coupled receptor (GPCR). For example, when bred to a strain expressing tTA in forebrain neurons (Camk2a-tTA; see Stock No. 003010), these transgenic mice are a model allowing in vivo chemical control of neuronal activity, neuronal firing, and non-neuronal signaling.

In addition, breeding TRE-hM3Dq mice with cfos-htTA mice (Stock No. 018306) generates double transgenic offspring with hM3Dq expression in excitatory neurons that are sufficiently active to drive the c-fos promoter. Those hM₃D_q^fos double transgenic mice allow CNO ligand-induced, artificial reactivation of neurons associated with a specific memory. Temporal modulation may also be employed using dox. Such hM₃D_q^fos double transgenic mice allow one to generate and artificially-activate a synthetic memory trace, and may be useful in studying the spatial pattern of activity at the time of learning and retrieval.

Of note, several chemogenetic/pharmacogenetic tool strains are available from The Jackson Laboratory Repository; including these Tet-responsive TRE-hM3Dq transgenic mice (Stock No. 014093),
the adora2A-rM3Ds transgenic mice (Stock No. 017863),
the Tet-responsive TRE-hM4Di transgenic mice (Stock No. 024114),
the Cre-inducible R26-LSL-Gi-DREADD mice (R26-hM4Di/mCitrine; Stock No. 026219)
and the Cre-inducible CAG-LSL-Gq-DREADD mice (Tg-hM3Dq/mCitrine [formerly R26-LSL-Gq-DREADD]; Stock No. 026220).

For CNO protocol, see the detailed protocol for dissolving CNO obtained from the attachments section of the Designer Receptors Exclusively Activated by Designer Drugs DREADD wiki webpage.

Development

The TRE-HA-hM3Dq transgene was designed with a modified Tet response element (TRE or (tetO); described in detail below), a hemagglutinin epitope tag (HA), the hM3Dq sequence, and an SV40 polyA signal. The hM3Dq sequence is a G_q-coupled human M3 muscarinic DREADD (designer receptor exclusively activated by designer drug). To create the hM3Dq sequence, the wildtype human muscarinic 3 receptor (CHRM3) sequence was modified via site-directed mutagenesis to harbor two amino acid substitutions (Y149C^3.33/A239G^5.46) that abolish receptor affinity for the native ligand, acetylcholine (ACh), but allow receptor binding and subsequent activation by the small drug-like molecule clozapine-N-oxide (CNO).
This 2.36 kb TRE-HA-hM3Dq transgene was injected into the pronuclei of B6SJL hybrid mouse oocytes. Founder TRE-hM3Dq mice (line 1) were bred with B6;CBA-Tg(Camk2a-tTA)1Mmay/J (see Stock No. 003010) to establish the colony. TRE-hM3Dq transgenic mice were bred together or to wildtype (noncarrier) mice from the colony for approximately eight generations, and then crossed with C57BL/6J mice for one or two generations. The Camk2a-tTA transgene was removed during these breedings. Upon arrival at The Jackson Laboratory Repository, TRE-hM3Dq transgenic mice were bred to C57BL/6J inbred mice (Stock No. 000664) for at least one generation to establish the colony.

Additional information on hM3Dq:

More information on hM3Dq or other DREADDs may be available at the University of North Carolina Designer Receptors Exclusively Activated by Designer Drugs DREADD wiki webpage.

Additional information on TRE promoter:

The TRE promoter used in these transgenic mice is the Tet-responsive P_tight promoter. This P_tight promoter contains a modified Tet response element (TRE_mod), which consists of seven direct repeats of a 36-bp sequence each containing the 19-bp tet operator sequence (tetO). The TRE_mod is just upstream of a minimal human cytomegalovirus (CMV) promoter (P_minCMVΔ), which lacks the enhancer that is part of the complete CMV promoter. Consequently, P_tight is silent in the absence of binding of TetR or rTetR to the tetO sequences.

Expression Data

Expressed Gene	CHRM3, cholinergic receptor muscarinic 3, human
Site of Expression

Control Suggestions

Approximate Controls

100011 B6CBAF1/J, ()

Additional Information

Considerations for Choosing Controls

Selected References

Alexander GM; Rogan SC; Abbas AI; Armbruster BN; Pei Y; Allen JA; Nonneman RJ; Hartmann J; Moy SS; Nicolelis MA; McNamara JO; Roth BL. 2009. Remote control of neuronal activity in transgenic mice expressing evolved G protein-coupled receptors. Neuron 63(1):27-39PubMed: 19607790 MGI: J:154952

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Genetics

Tg(tetO-CHRM3*)1Blr

Allele Symbol: Tg(tetO-CHRM3*)1Blr

Allele Name	transgene insertion 1, Bryan Roth
Allele Type	Transgenic (Inducible, Inserted expressed sequence)
Allele Synonym(s)	Tg(tetO-CHRM3*)1Blr; transgene insertion 1, Bryan Roth
Gene Symbol and Name	Tg(tetO-CHRM3*)1Blr, transgene insertion 1, Bryan Roth
Gene Synonym(s)
Promoter	CMV, cytomegalovirus, human
Promoter	tetO, tet operator,
Expressed Gene	CHRM3, cholinergic receptor muscarinic 3, human
Strain of Origin	(C57BL/6 x SJL)F1
Chromosome	UN
Molecular Note	The Tet-responsive P promoter which consists of seven direct repeats of 36-bp sequence each containing the 19-bp tet operator sequence (tetO) is just upstream of a minimal human cytomegalovirus (CMV) promoter. This is upstream of a hemagglutinin epitop tag and a Gq-coupled human M3 muscarinic receptor. The wildtype human muscarinic 3 receptor sequence was modified via site-directed mutagenesis to harbor two amino acid substitutions (Y149C and A239G) that abolish receptor affinity for the native ligand, acetylcholine (ACh), but allow receptor binding and subsequent activation by the small drug-like molecule clozapine-N-oxide (CNO).
Mutations Made By	Bryan Roth, University of North Carolina at Chapel Hill

Disease/Phenotype

Disease Terms

Research Areas By Genotype

This mouse can be used to support research in many areas including:

Research Tools
- Cardiovascular Research
  - Tetop Tet System
- Genetics Research
  - Mutagenesis and Transgenesis: Tetop Tet System
- Neurobiology Research
  - Tetop Tet System
- Reproductive Biology Research
  - Tetop Tet System
- Tet Expression Systems
  - tTA/rtTA Responsive Strains
- Cancer Research
  - Tetop Tet System
Neurobiology Research
- Optogenetic and Chemogenetic tools
  - Chemogenetic tools
- Receptor Defects
  - G protein-coupled receptor
- Tet Expression System
  - tTA/rtTA Responsive Strains
Diabetes and Obesity Research
- Insulin Receptors and Growth Factors
Immunology, Inflammation and Autoimmunity Research
- Growth Factors/Receptors/Cytokines
- Intracellular Signaling Molecules
Cell Biology Research
- Signal Transduction
Cancer Research
- Growth Factors/Receptors/Cytokines

Mammalian Phenotype Terms by Genotype

References

Alexander GM; Rogan SC; Abbas AI; Armbruster BN; Pei Y; Allen JA; Nonneman RJ; Hartmann J; Moy SS; Nicolelis MA; McNamara JO; Roth BL. 2009. Remote control of neuronal activity in transgenic mice expressing evolved G protein-coupled receptors. Neuron 63(1):27-39PubMed: 19607790 MGI: J:154952

Additional - Tg(tetO-CHRM3*)1Blr related

Technical Support

Contact Technical Support

Genotyping Protocols
- Pyrosequencing: ** human CHRM3*Y149C
- Pyrosequencing: ** human CHRM3*A239G
- Separated PCR: Tg(tetO-CHRM3*)1Blr
- Standard PCR: Tg(tetO-CHRM3*)1Blr
- High Resolution Melting: Tg(tetO-CHRM3*)1Blr
- Genotyping resources and troubleshooting
Breeding Considerations

When maintaining a live colony, transgene carrier mice may be bred together or to wildtype (noncarrier) mice from the colony. The donating investigator reports they have not tried to generate homozygous mice (November 2011).
- Additional Breeding and Husbandry Support
Citation
When using the TRE-hM3Dq mouse strain in a publication, please cite the originating article(s) and include JAX stock #014093 in your Materials and Methods section.

Animal Health Reports

Facility Barrier Level Descriptions

Production of mice from cryopreserved embryos or sperm occurs in a maximum barrier room, G200

Pricing & Availability

Cryo Recovery

Domestic
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Live Mouse

Age

Genotype

Price

weeks

Cryorecovery - Pricing

Service	Genotype	Price
	Hemizygous or Non carrier for Tg(tetO-CHRM3*)1Blr

We will fulfill your order by providing at least two carriers for each strain ordered. The total number, sex, and genotypes provided will vary, although typically 8 or more animals are provided. Please check genotypes which will be recovered. While the genotypes of all animals produced will be communicated to you prior to scheduling shipment, the genotypes of animals provided may not reflect the mating scheme and genotypes described in the strain description. Animals are typically ready to ship in 11-14 weeks. If a second recovery is required to produce the minimum number of animals, then delivery time would increase to approximately 25 weeks. If we fail to produce animals of the correct genotype, you will not be charged. We cannot guarantee the reproductive success of mice shipped to your facility. If the mice are lost after the first three days (post-arrival) or do not produce progeny at your facility, a new order and fee will be necessary.

Cryorecovery to establish a Dedicated Supply for greater quantities of mice. Mice recovered can be used to establish a dedicated colony to contractually supply you mice according to your requirements. Price by quotation.

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Also Known As: TRE-hM3Dq

Donating Investigator

Genetic overview

Research Applications

Base Price

Detailed Description

Development

Expression Data

Control Suggestions

Approximate Controls

Additional Information

Selected References

Tg(tetO-CHRM3*)1Blr

Allele Symbol: Tg(tetO-CHRM3*)1Blr

Disease Terms

Research Areas By Genotype

This mouse can be used to support research in many areas including:

Mammalian Phenotype Terms by Genotype

References

Additional - Tg(tetO-CHRM3*)1Blr related

Genotyping Protocols

Breeding Considerations

Citation

Animal Health Reports

Production of mice from cryopreserved embryos or sperm occurs in a maximum barrier room, G200

Live Mouse

Cryorecovery - Pricing

Related Products and Services

Payment Terms and Conditions

The Jackson Laboratory's Genotype Promise

Terms of Use

Additional Use Restrictions Apply

Licensing Information

JAX® Mice, Products & Services Conditions of Use

No Warranty

Credit for PRODUCTS or SERVICES

Animal Care and Use for SERVICES

No Liability